NOTE: The entire reaction can be used for a transformation without any further purification.

MATERIALS

10X Taq Buffer: 0.5 M KCl 100 mM Tris-Cl, pH 8.5 1% Triton X-100

25 mM MgCl2

10 mM dNTP's

pRS40X template DNA - mini-prep DNA works well

Taq polymerase

Two Gene-specific DNA primers: One oligonucleotide should consist of 40 nts of gene-specific sequence for one end of the targeted region at the 5' end followed by: 5'-CTGTGCGGTATTTCACACCG-3' (left primer), and another 40 nt homologous to the other side of the targeted region at the 5' end followed by: 5' AGATTGTACTGAGAGTGCAC-3' (right primer). The primers are then used to amplify any auxotrophic marker from a pRS40X or pRS30X integrating plasmid (Sikorski & Hieter, 1989; Brachmann et al. 1998).